Protein/amino acid availability is a critical factor in general nutrition in mammals, yet there is relatively little known about the impact of amino acid fluctuation on cell functions, such as transcription. We use amino acid deprivation of cultured cells to investigate transcriptional control by nutrients. Limiting the amount of any single amino acid suppresses general protein synthesis, but actually promotes increased translation of a subset of mRNAs, including the transcription factor ATF4. Our hypothesis is that ATF4 is a critical mediator of the amino acid stress response pathway and that the human asparagine synthetase (ASNS) gene is a model for the study of this pathway. Our previous work has documented ATF4-mediated activation of the ASNS gene through the promoter cis-element Nutrient Sensing Response Element-1 (NSRE-1). In association with enhancer binding, a "pre-initiation complex" is assembled at eukaryotic promoters, which is comprised of at least four multi-protein complexes: a chromatin remodeling complex, a Mediator complex, a general transcription factor complex, and the RNA Pol II holoenzyme. The role of ATF4 and its sub-domains in factor recruitment to the ASNS promoter will be studied by chromatin immunoprecipitation (ChIP) in ATF4-/- fibroblasts and ATF4-siRNA treated HepG2 hepatoma cells (Specific Aim I). ATF4 binding proteins will be identified by yeast 2-hybrid and studied by co-IP, GST pull-down, and ChIP (Specific Aim II).To identify other proteins at the ASNS promoter, ATF4-containing complexes will be isolated, individual proteins identified by mass spectrometry, and the ATF4-interacting proteins studied by co-IP and ChIP (Specific Aim III). These studies will yield valuable information about the mechanisms by which nutritional signals mobilize the transcriptional machinery and thereby, alter cellular metabolism through gene expression. Given the central role of ATF4 in the response to amino acid limitation, and other forms of nutritional stress, we believe that it is critical to understand its function in the assembly of the general transcriptional machinery.